miR-889-3p靶向Runx2抑制脂肪间充质干细胞的成骨分化

miR-889-3p inhibits osteogenic differentiation of adipose-derived mesenchymal stem cells by targeting Runx2

背景:研究表明miR-889-3p可参与调控多种细胞的增殖分化过程,但是其对脂肪间充质干细胞成骨分化的影响尚不明确。目的:探讨miR-889-3p对脂肪间充质干细胞成骨分化的调节作用。方法:体外分离、培养SD大鼠脂肪间充质干细胞,转染miR-889-3p模拟物(miR-889-3p mimic)、miR-889-3p抑制剂(miR-889-3p inhibitor)和阴性对照(miR-NC)并诱导成骨分化。RT-PCR和Western blot检测成骨诱导14 d后的碱性磷酸酶活性、Runx2、骨钙素、骨桥素、Osterix等成骨相关mRNA和蛋白的表达。将野生型Runx2、突变型Runx2分别与miR-889-3p模拟物和miR-NC共转染脂肪间充质干细胞,通过双荧光素酶报告基因检测miR-889-3p与Runx2的靶向关系。结果与结论:①miR-889-3p表达水平随成骨诱导时间延长而逐渐减低;②成骨诱导第7天和第14天miR-889-3p inhibitor组的矿化结节数目、大小、颜色深浅明显超过miR-889-3p mimic组和miR-NC组(P<0.05);③miR-889-3p过表达后,碱性磷酸酶活性、Runx2、骨钙素、骨桥素、Osterix mRNA及蛋白表达量显著降低,而miR-889-3p敲除后成骨相关mRNA和蛋白表达量明显提高;④双荧光素酶报告基因检测结果显示miR-889-3p过表达显著降低了野生型Runx2的荧光素酶活性;⑤上述结果表明,miR-889-3p通过靶向Runx2负向调控脂肪间充质干细胞成骨分化。

BACKGROUND:Studies have shown that miR-889-3p is able to participate in the regulation of the proliferation and differentiation of a variety of cells,but its effect on the osteogenic differentiation of adipose-derived mesenchymal stem cells remains unclear.OBJECTIVE:To investigate the regulation of miR-889-3p in osteogenic differentiation of adipose-derived mesenchymal stem cells.METHODS:Adipose-derived mesenchymal stem cells from SD rats were isolated and cultured in vitro,transfected with miR-889-3p mimics,Mir-889-3p inhibitor and negative control(miR-NC)then induced osteogenic differentiation.RT-PCR and western blot assay were used to detect alkaline phosphatase activity,Runx2,osteocalcin,osteopontin,Osterix and other osteogenic related mRNA and protein expression levels.Adipose-derived mesenchymal stem cells were co-transfected with wild-type Runx2 and mutant Runx2 with miR-889-3p mimic and miR-NC,respectively.Luciferase reporter gene assay was used to detect the targeting relationship between miR-889-3p and Runx2.RESULTS AND CONCLUSION:(1)The expression level of miR-889-3p decreased gradually with the prolongation of osteogenic induction.(2)The number,size,and color depth of mineralized nodules in the miR-889-3p inhibitor group were significantly higher than those in the miR-889-3p mimic group and miR-NC group on days 7 and 14 of osteogenic induction(P<0.05).(3)Overexpression of miR-889-3p significantly suppressed alkaline phosphatase activity,Runx2,osteocalcin,osteopontin,Osterix mRNA and protein expression,whereas the knockdown of miR-889-3p remarkably enhanced osteogenesis-related mRNA and protein expression.(4)The results of dual-luciferase reporter gene assay showed that overexpression of miR-889-3p significantly reduced the luciferase activity of wild-type Runx2.(5)These results suggest that miR-889-3p negatively regulates osteogenic differentiation of adipose-derived mesenchymal stem cells by targeting Runx2.